Anionic rhamnolipids are known to be formed by growing submersed cultures of microorganisms, in particular of the genus Pseudomonas.
Thus for example in the culture of Pseudomonas pyocyanea in a casein/glucose medium according to the method described by Bergstrom et al in Arkiv for Kemi, Mineralogi och Geologi, Vol. 23 A (1946) No. 13, pp. 1 to 12, a "pyolipic acid" is formed which is referred to as acidic lipid.
Jarvis et al in J. Amer. Chem. Soc. 71 (1949), pp. 4124 to 4126 describe an anionic rhamnolipid-(I), which is designated 2-0-.alpha.-L-rhamnopyranosyl-.alpha.-L-rhamnopyranosyl-.beta.-hydroxydeca noyl-.beta.-hydroxydecanoic acid and accordingly has the following structural formula: ##STR1##
Hisatsuka et al in Agr. Biol. Chem., Vol. 5 (1971), No. 5, pp. 686 to 692 describe the formation of rhamnolipids by Pseudomonas aeruginosa and their effect in the fermentation of hydrocarbons. These authors obtained rhamnolipid (I) with Pseudomonas aeruginosa using n-paraffins as carbon and energy source. This rhamnolipid (I) was found to promote the growth of Pseudomonas aeruginosa in an n-hexadecane medium to a very pronounced degree even in relatively small concentrations. This growth-promoting effect observed here with rhamnolipid (I) could be detected in other microorganisms which degrade hydrocarbons.
Itoh and co-workers, in their publication in The Journ. of Antibiotics, Vol. 24 (1971), pp. 855 to 859 take this state of the art as their starting point and state that the more precise knowledge of the biosynthesis of rhamnolipid (I) gives grounds to suppose that yet another rhamnolipid is formed which is a precursor of rhamnolipid (I). These investigators found that, from a culture of Pseudomonas aeruginosa growing on n-paraffin, not only the known rhamnolipid (I), which they designated rhamnolipid R-1, but also a further rhamnolipid R-2 could be isolated, which could be identified as L-.alpha.-rhamnopyranosyl-.beta.-hydroxydecanoyl-.beta.-hydroxydecanoate and which was recognized as a precursor of rhamnolipid (I).
These findings are also presented, using the same definitions, by Itoh et al in Agr. Biol. Chem., Vol. 36 (1972), pp. 2233 to 2235. They also report that mutants of Pseudomonas aeruginosa which have lost the ability to use n-paraffin as nutrient grow normally on n-paraffins when rhamnolipids are added to the culture suspension. From this they conclude that the mutants have not lost the enzymatic activity which enables them to use n-paraffins as a nutrient source and that the rhamnolipids are indispensable for the degradation of n-paraffins by Pseudomonas aeruginosa microorganisms.
Cooper et al, in a report on rhamnolipids in Microbial Degradation of Pollutants in Marine Environments (1979), pp. 231 to 240, refer to the aforementioned papers and reproduce the designation of the rhamnolipid verbatim in agreement with Itoh et al loc. cit. (1971). However, for the rhamnolipid referred to as .alpha.-L-rhamnopyranosyl-.beta.-hydroxydecanoyl-.beta.-hydroxydecanoate these authors incorrectly state a structural formula which exhibits only one rhamnose residue and also only one hydroxydecanoic acid residue. The same error can also be found in the publication of Cooper et al in Advances in Applied Microbiol., Vol. 26 (1980), pp. 229 to 253, on pp. 234 and 235.
From DE-OS No. 21 50 375, a method for the biotechnical production of rhamnose-containing glycolipids is known, in which a similar glycolipid-producing bacterium of the genus Pseudomonas is cultured aerobically in an aqueous nutrient medium containing at least one assimilable carbon source at pH levels of 4 to 10 and temperatures of 25.degree. to 37.degree. C. and in which the glycolipids are isolated from the nutrient broth. This process was designed to allow glycolipids to be obtained in high yields. According to the examples given in this DE-OS, the following yields are obtained with the stated Pseudomonas species on the C-base assigned to them:
______________________________________ rhamnolipid yield in Pseudomonas species carbon source g/l culture broth ______________________________________ aeruginosa ATCC 15246 n-paraffin 2.4 fluorescens ATCC 15453 n-paraffin 5.0 aeruginosa ATCC 10145 glycerin 0.87 aeruginosa ATCC 10145 n-paraffin 9.7 aeruginosa ATCC 15246 glucose 5.6 fluorescens ATCC 15453 glycerin 3.3 ______________________________________
As rhamnolipids, .alpha.-L-rhamnopyranosyl-.beta.-hydroxydecanoyl-.beta.-hydroxydecanoic acid and 2-0-L-rhamnopyranosyl-.alpha.-L-rhamnopyranosyl-.beta.-hydroxydecanoic acid are obtained.
From this state of the art it is apparent that to date it has only been possible to produce rhamnolipids biotechnically which contain 2 hydroxydecanoic acid residues together with one or two rhamnose residues.
The object of the present invention is to discover addition rhamnolipids and means of increasing the yield of rhamnolipids relative to the cell substance.